Bacterial Genetics and Gene Cloning Summary

Prokaryotes: Small in size (0.5 - 5 µm), lack organelles, DNA in nucleoid (no nucleus), reproduce by binary fission.
Chromosomal DNA:
- Circular in shape, length of a few million base pairs.
- Single type, may have multiple copies; several thousand genes present.
- One origin of replication is required.
Plasmid DNA:
- Small circular molecules, 3000-500,000 bp, contains several dozen to hundreds of genes.
- Each plasmid has its own origin of replication.
Binary Fission:
- Process where DNA replicates, new cell wall forms, and cell divides into two identical daughter cells.
Gene Transfer Modes:
- Conjugation: Direct transfer of DNA.
- Transformation: Uptake of naked DNA from the environment.
- Transduction: Virus-mediated transfer of DNA.

Gene Cloning: Producing large copies of a specific DNA sequence.
Vector: DNA molecule used to carry the gene of interest (e.g., plasmids, viruses).
Gene of Interest: The targeted DNA sequence to clone.
Cloning Steps:
1. Isolate vector: Extract vector DNA and gene from the chromosomal DNA.
2. Insert gene: Combine gene of interest into vector DNA using restriction enzymes.
3. Introduce recombinant vector: Place it into a host cell; it replicates during cell division.
4. Transform bacteria: Make bacterial cells competent to uptake plasmid DNA.
5. Plate cells onto selective media: Use antibiotics to select for transformed cells.
6. Identify desired clone: Techniques like colony hybridization, Southern blotting, and PCR.

Components: Template DNA, Taq polymerase, nucleotides, primers, buffers.
Temperature Cycling:
- 95°C: Melting DNA strands.
- 65°C: Annealing primers.
- 72°C: Polymerizing new strands.
Multiplication: From 1 copy to millions after ~30 cycles.

Pour agarose gel, include salts and dyes.
Load DNA into wells and apply electrical field.
DNA moves towards the positive electrode; smaller fragments travel faster than larger ones.